Variation in endocrine signaling underlies variation in social life history.

Variation in endocrine pathways can be a major mechanism underlying life-history evolution. Yet it is unclear whether this insight, derived primarily from solitary species, explains the origins of complex life-history traits in highly social taxa. Thus, we here document and study variation in social life-history syndromes of female fecundity, behavior, and life span in selectively bred honeybee (Apis mellifera) strains. Associated variation in endocrine signaling was uncovered by RNA interference (RNAi) silencing of the juvenile hormone (JH) suppressor gene vitellogenin. High versus low endocrine reactivity in response to vitellogenin knockdown consistently correlated with rapid social behavioral ontogeny and short life span versus slow social behavioral ontogeny and long life span. Variation in JH reactivity, furthermore, was a function of variation in fecundity (ovary size and follicle development). A JH-mediated pleiotropy of female life-history traits, including fecundity, behavior, and life span, characterizes the distantly related solitary insect Drosophila. For the first time, we document a similar regulatory principle in a highly social species where most females are alloparental helpers (workers) that seldom reproduce. We conclude that variation in endocrine pathways of solitary origin can underlie variation and evolvability of complex social life-history traits.

Immunogold localization of vitellogenin in the ovaries, hypopharyngeal glands and head fat bodies of honeybee workers, Apis mellifera.

Vitellogenin is a yolk precursor protein in most oviparous females. In the advanced eusocial honeybee, Apis mellifera (Hymenoptera: Apidae), vitellogenin has recently attracted much interest as this protein, in addition to a classical function in oocyte development in the reproductive queen caste, has evolved functions in the facultatively sterile female worker caste not documented in other species. However, research on the spatial dynamics of vitellogenin in various tissues is not easily performed with available tools. Here we present an immunogold staining procedure that visualizes honeybee vitellogenin in resin embedded tissue. To establish the protocol, we used ovaries of worker bees from colonies with and without a queen. Under the first condition, vitellogenin is assumed not to be present in the workers' ovaries. Under the second condition, the ovaries of worker bees become vitellogenic, with abundant opportunities for detection of complex patterns of vitellogenin uptake and storage. By use of this experimental setup, the staining method is shown to be both sensitive and specific. To demonstrate the functional significance of the protocol, it was subsequently used to identify vitellogenin protein in the hypopharyngeal glands (brood food producing head glands) of nursing worker bees and in adjacent head fat body cells for the first time. Localization of vitellogenin in these tissues supports previously hypothesized roles of vitellogenin in social behavior. This protocol thus provides deeper insights into the functions of vitellogenin in the honeybee.

Long-term maintenance of in vitro cultured honeybee (Apis mellifera) embryonic cells.

BACKGROUND: In vitro cultivation of cells allows novel investigation of in vivo- mechanisms and is a helpful tool in developmental biology, biochemistry and functional genomics. Numerous cell lines of insect species, e.g., silkworm and mosquito, have been reported. However, this is not the case for successful long-term cultivation of cells in honeybees. RESULTS: Methods for cultivation of honeybee embryonic cells are discussed here. Pre-gastrula stage embryos were used to initiate cultures, and cells were reared on 96-wells microplates with Grace insect medium, supplemented with Fetal Bovine Serum. Cells proliferated in clusters, and maintained viable and mitotic for more than three months. CONCLUSION: We report here, for the first time, long-term cultivation of honeybee cells. Results represent a highly useful in vitro-system for studying a model organism of increasing importance in areas such as aging, sociality and neurobiology.

Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera).

In the honey bee (Apis mellifera), young workers usually perform tasks in the nest while older workers forage in the field. The behavioral shift from nest-task to foraging activity is accompanied by physiological and sensory changes so that foragers can be characterized by a higher juvenile hormone (JH) level, a lower vitellogenin protein titer, and an increased responsiveness to water and sucrose stimuli. JH was hypothesized to be the key mediator of behavioral development, physiology, and sensory sensitivity in honey bee workers. Recent research, however, has shown that JH is controlled by the hemolymph vitellogenin level, which implies that the fat body specific vitellogenin gene can be a key regulator of behavioral change. Here, we show that downregulation of vitellogenin activity by RNA interference (RNAi) causes an increase in the gustatory responsiveness of worker bees. Our observations suggest that vitellogenin is an important regulator of long-term changes in honey bee behavior.

Social reversal of immunosenescence in honey bee workers.

A striking example of immunosenescence is seen in the honey bee (Apis mellifera) worker caste. The bees' age-associated transition from hive duties to more risky foraging activities is linked to a dramatic decline in immunity. Explicitly, it has been shown that an increase in the juvenile hormone (JH) level, which accompanies onset of foraging behavior, induces extensive hemocyte death through nuclear pycnosis. Here, we demonstrate that foragers that are forced to revert to hive-tasks show reversal of immunosenescence, i.e. a recovery of immunity with age. This recovery, which is triggered by a social manipulation, is accompanied by a drop in the endogenous JH titer and an increase in the hemolymph vitellogenin level. Vitellogenin is a zinc binding glycolipoprotein that has been implicated in the regulation of honey bee immune integrity. We also establish that worker immunosenescence is mediated by apoptosis, corroborating that reversal of immunosenescence emerges through proliferation of new cells. The results presented here, consequently, reveal a unique flexibility in honey bee immunity--a regulatory plasticity that may be of general biological interest.

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA.

BACKGROUND: The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees. RESULTS: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96%) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days. CONCLUSION: Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

Hormonal control of the yolk precursor vitellogenin regulates immune function and longevity in honeybees.

A striking example of plasticity in life span is seen in social insects such as ants and bees, where different castes may display distinct ageing patterns. In particular, the honeybee offers an intriguing illustration of environmental control on ageing rate. Honeybee workers display a temporal division of labour where young bees (or 'hive bees') perform tasks within the brood nest, and older bees forage for nectar, pollen propolis and water. When bees switch from the hive bee to the forager stage, their cellular defence machinery is down-regulated by a dramatic reduction in the number of functioning haemocytes (immunocytes). This study documents that the yolk precursor vitellogenin is likely to be involved in a regulatory pathway that controls the observed decline in somatic maintenance function of honeybee foragers. An association between the glyco-lipoprotein vitellogenin and immune function has not previously been reported for any organism. Honeybee workers are functionally sterile, and via the expression of juvenile hormone, a key gonotrophic hormone in adult insects, their vitellogenin levels are influenced by social interactions with other bees. Our results therefore suggest that in terms of maintenance of the cellular immune system, senescence of the honeybee worker is under social control.

Altered physiology in worker honey bees (Hymenoptera: Apidae) infested with the mite Varroa destructor (Acari: Varroidae): a factor in colony loss during overwintering?

The ectoparasitic mite Varroa destructor (Anderson & Trueman) is the most destructive pest of the honey bee, Apis mellifera L., in Europe and the United States. In temperate zones, the main losses of colonies from the mites occur during colony overwintering. To obtain a deeper knowledge of this phenomenon, we studied the mites' impact on the vitellogenin titer, the total protein stores in the hemolymph, the hemocyte characteristics, and the ecdysteroid titer of adult honey bees. These physiological characteristics are indicators of long-time survival and endocrine function, and we show that they change if bees have been infested by mites during the pupal stage. Compared with noninfested workers, adult bees infested as pupae do not fully develop physiological features typical of long-lived wintering bees. Management procedures designed to kill V. destructor in late autumn may thus fail to prevent losses of colonies because many of the adult bees are no longer able to survive until spring. Beekeepers in temperate climates should therefore combine late autumn management strategies with treatment protocols that keep the mite population at low levels before and during the period when the winter bees emerge.

A diapause pathway underlies the gyne phenotype in Polistes wasps, revealing an evolutionary route to caste-containing insect societies.

Colonies of social wasps, ants, and bees are characterized by the production of two phenotypes of female offspring, workers that remain at their natal nest and nonworkers that are potential colony reproductives of the next generation. The phenotype difference includes morphology and is fixed during larval development in ants, honey bees, and some social wasps, all of which represent an advanced state of sociality. Paper wasps (Polistes) lack morphological castes and are thought to more closely resemble an ancestral state of sociality wherein the phenotype difference between workers and nonworkers is established only during adult life. We address an alternative hypothesis: a bias toward the potential reproductive (gyne) phenotype among Polistes female offspring occurs during larval development and is based on a facultatively expressed ancestral life history trait: diapause. We show that two signatures of diapause (extended maturation time and enhanced synthesis and sequestration of a hexameric storage protein) characterize the development of gyne offspring in Polistes metricus. Hexameric storage proteins are implicated in silencing juvenile hormone signaling, which is a prerequisite for diapause. Diverging hexamerin protein dynamics driven by changes in larval provisioning levels thereby provide one possible mechanism that can cause an adaptive shift in phenotype bias during the Polistes colony cycle. This ontogenetic basis for alternative female phenotypes in Polistes challenges the view that workers and gynes represent behavior options equally available to every female offspring, and it exemplifies how social insect castes can evolve from casteless lineages.

Reproductive protein protects functionally sterile honey bee workers from oxidative stress.

Research on aging shows that regulatory pathways of fertility and senescence are closely interlinked. However, evolutionary theories on social species propose that lifelong care for offspring can shape the course of senescence beyond the restricted context of reproductive capability. These observations suggest that control circuits of aging are remodeled in social organisms with continuing care for offspring. Here, we studied a circuit of aging in the honey bee (Apis mellifera). The bee is characterized by the presence of a long-lived reproductive queen caste and a shorter-lived caste of female workers that are life-long alloparental care givers. We focus on the role of the conserved yolk precursor gene vitellogenin that, in Caenorhabditis elegans, shortens lifespan as a downstream element of the insulin/insulin-like growth factor signaling cascade. Vitellogenin protein is synthesized at high levels in honey bee queens and is abundant in long-lived workers. We establish that vitellogenin gene activity protects worker bees from oxidative stress. Our finding suggests that one mechanistic explanation for patterns of longevity in bees is that a reproductive regulatory pathway has been remodeled to extend life. This perspective is of considerable relevance to research on longevity regulation that builds largely on inference from solitary model species.

Social exploitation of vitellogenin.

Vitellogenin is a female-specific glucolipoprotein yolk precursor produced by all oviparous animals. Vitellogenin expression is under hormonal control, and the protein is generally synthesized directly before yolk deposition. In the honeybee (Apis mellifera), vitellogenin is not only synthesized by the reproductive queen, but also by the functionally sterile workers. In summer, the worker population consists of a hive bee group performing a multitude of tasks including nursing inside the nest, and a forager group specialized in collecting nectar, pollen, water, and propolis. Vitellogenin is synthesized in large quantities by hive bees. When hive bees develop into foragers, their juvenile hormone titers increase, and this causes cessation of their vitellogenin production. This inverse relationship between vitellogenin synthesis and juvenile hormone is opposite to the norm in insects, and the underlying proximate processes and life-history reasons are still not understood. Here we document an alternative use of vitellogenin by showing that it is a source for the proteinaceous royal jelly that is produced by the hive bees. Hive bees use the jelly to feed larvae, queen, workers, and drones. This finding suggests that the evolution of a brood-rearing worker class and a specialized forager class in an advanced eusocial insect society has been directed by an alternative utilization of yolk protein.

Reproductive ground plan may mediate colony-level selection effects on individual foraging behavior in honey bees.

The colony-level phenotype of an insect society emerges from interactions between large numbers of individuals that may differ considerably in their morphology, physiology, and behavior. The proximate and ultimate mechanisms that allow this complex integrated system to form are not fully known, and understanding the evolution of social life strategies is a major topic in systems biology. In solitary insects, behavior, sensory tuning, and reproductive physiology are linked. These associations are controlled in part by pleiotropic networks that organize the sequential expression of phases in the reproductive cycle. Here we explore whether similar associations give rise to different behavioral phenotypes in a eusocial worker caste. We document that the pleiotropic genetic network that controls foraging behavior in functionally sterile honey bee workers (Apis mellifera) has a reproductive component. Associations between behavior, physiology, and sensory tuning in workers with different foraging strategies indicate that the underlying genetic architectures were designed to control a reproductive cycle. Genetic circuits that make up the regulatory "ground plan" of a reproductive strategy may provide powerful building blocks for social life. We suggest that exploitation of this ground plan plays a fundamental role in the evolution of social insect societies.